SGX70393 inhibits the T315I mutant of Bcr-Abl kinase and pre-empts drug resistance when combined with nilotinib or dasatinib

A259 Overview: Bcr-AblT315I is resistant to all currently available kinase inhibitors. We report that SGX70393 is a potent, azapyridine-based inhibitor of native and T315I-mutant Bcr-Abl kinase. Furthermore, combining SGX70393 with nilotinib or dasatinib completely abrogates emergence of resistant subclones, including Bcr-AblT315I, in a cell-based screen. Methods: We assessed the efficacy of SGX70393 in cellular and biochemical assays against a panel of Bcr-Abl kinase domain (KD) mutants: (M244V, G250E, Q252H, Y253F, Y253H, E255K, E255V, F311L, T315A, T315I, F317L, M351T, F359V, V379I, L387M, H396P, or H396R). Colony formation assays were carried out with murine bone marrow cells expressing Bcr-Abl or Bcr-AblT315I and toxicity was assessed in colony forming assays of normal human mononuclear cells. SGX70393 was also evaluated in a xenograft mouse model using Ba/F3 Bcr-AblT315I cells and in cellular tyrosine phosphorylation assays with primary human Bcr-AblT315I cells. The resistance profile of SGX70393 was also evaluated alone and in combination with imatinib, nilotinib, or dasatinib using a recently developed accelerated, cell-based mutagenesis assay [Bradeen, et al. Blood (2006) 108:2232-38]. Results:SGX70393 inhibited growth of cells expressing Bcr-AblT315I (IC50: 7.3 nM) or native Bcr-Abl (IC50: 12 nM). Sensitivity of Bcr-Abl mutants to SGX70393 partitioned into three categories: high (IC50 500 nM: G250E, Y253F, E255V, F317L). Colony formation by murine bone marrow cells expressing Bcr-AblT315I was reduced by SGX70393 while no toxicity was observed in colony forming assays of normal human or murine mononuclear cells treated with SGX70393. A cell-based mutagenesis screen for Bcr-Abl kinase domain escape mutants emerging in the presence of SGX70393 revealed that four previously reported Bcr-Abl mutations account for almost all resistance. Results from experiments using SGX70393 in combination with imatinib, nilotinib and dasatinib suggest significant further reductions of resistant clones. Remarkably, outgrowth of resistant clones was completely suppressed when SGX70393 was used in combination with nilotinib or dasatinib. Conclusions:SGX70393 is a potent inhibitor of native and T315I mutant Bcr-Abl, with coverage extending to most clinically relevant mutants except those in the P-loop and F317. Outgrowth of Bcr-AblT315I is reported to be a common mechanism of resistance to dasatinib and nilotinib. As such, an effective Bcr-AblT315I inhibitor will be necessary to re-establish response in these relapsed patients. In addition, a Bcr-AblT315I inhibitor is likely to become a critical component of Abl kinase inhibitor combinations administered up-front to delay emergence of drug resistance in patients with advanced disease. Our pre-clinical findings suggest that SGX70393 in dual combination with nilotinib or dasatinib has the potential to prevent outgrowth of any known escape mutant, thus eliminating kinase inhibitor resistance.